Skip to main content
SpringerLink
Log in

Quantification of Oligodendrocytes and Myelin in Human iPSC-Derived 3D Brain Cell Cultures (BrainSpheres)

  • Protocol
  • First Online:
Experimental Neurotoxicology Methods

Part of the book series: Neuromethods ((NM,volume 172))

Abstract

Myelination is considered a critical process in the development of the vertebrate brain. This process is susceptible and can be affected by exposure to developmental neurotoxicants or by numerous diseases (e.g., Schizophrenia, bipolar disorder, amyotrophic lateral sclerosis). Studying human myelination has been very difficult due to the lack of in vitro human models capable of reproducing this process. A human 3D iPSC-derived brain model (also called BrainSpheres—BS), developed by Johns Hopkins University, is a multicellular culture that includes different neuronal and glial cell types such as neurons, astrocytes, and oligodendrocytes, and is able to mimic human myelination in vitro. Here, we describe all the methods developed in this model in the last years to quantify oligodendrocytes and myelination. Application of Computer-assisted Evaluation of Myelin (CEM) (developed by Kertman et al.) and other immunochemistry quantification methods are here adapted to a 3D culture BrainSpheres.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 279.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Notes

  1. 1.

    http://www.biologists.com/DEV_Movies/DEV116517/DEV116517_Appendix%20S2-CEM%20package.zip

References

  1. Bunge MB, Pappas GD, Bunge RP (1962) Electron microscopic demonstration of connections between glia and myelin sheaths in developing mammalian central nervous system. J Cell Biol 12(2):448–453

    CAS  PubMed  Google Scholar 

  2. Branson HM (2013) Normal myelination: a practical pictorial review. Neuroimaging Clin N Am 23(2):183–195

    PubMed  Google Scholar 

  3. Guleria S, Kelly TG (2014) Myelin, myelination, and corresponding magnetic resonance imaging changes. Radiol Clin N Am 52(2):227–239

    PubMed  Google Scholar 

  4. Mathews ES, Appel B (2016) Oligodendrocyte differentiation. Zebrafish: cellular and developmental biology. Dev Biol 134:69–96

    CAS  Google Scholar 

  5. Laule C et al (2007) Magnetic resonance imaging of myelin. Neurotherapeutics 4(3):460–484

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Navel KA, Salzer JL (2006) Axonal regulation of myelination by neuregulin 1. Curr Opin Neurobiol 16(5):492–500

    Google Scholar 

  7. Podbielska M, Hogan EL (2009) Molecular and immunogenic features of myelin lipids: incitants or modulators of multiple sclerosis? Mult Scler J 15(9):1011–1029

    CAS  Google Scholar 

  8. van der Knaap MS, Wolf NI, Heine VM (2016) Leukodystrophies five new things. Neurol Clin Pract 6(6):506–514

    PubMed  PubMed Central  Google Scholar 

  9. Barkovich AJ (2000) Concepts of myelin and myelination in neuroradiology. Am J Neuroradiol 21(6):1099–1109

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Bergles DE et al (2000) Glutamatergic synapses on oligodendrocyte precursor cells in the hippocampus. Nature 405(6783):187–191

    CAS  PubMed  Google Scholar 

  11. Karadottir R et al (2008) Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter. Nat Neurosci 11(4):450–456

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Lin SC, Bergles DE (2004) Synaptic signaling between GABAergic interneurons and oligodendrocyte precursor cells in the hippocampus. Nat Neurosci 7(1):24–32

    CAS  PubMed  Google Scholar 

  13. Brady ST et al (1999) Formation of compact myelin is required for maturation of the axonal cytoskeleton. J Neurosci 19(17):7278–7288

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Funfschilling U et al (2012) Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity. Nature 485(7399):517–521

    PubMed  PubMed Central  Google Scholar 

  15. Lee YJ et al (2012) Oligodendroglia metabolically support axons and contribute to neurodegeneration. Nature 487(7408):443–U1502

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Miller RH (2002) Regulation of oligodendrocyte development in the vertebrate CNS. Prog Neurobiol 67(6):451–467

    CAS  PubMed  Google Scholar 

  17. Nave KA, Werner HB (2014) Myelination of the nervous system: mechanisms and functions. Ann Rev Cell Dev Biol 30:503–533

    CAS  Google Scholar 

  18. Saab AS, Tzvetanova ID, Nave KA (2013) The role of myelin and oligodendrocytes in axonal energy metabolism. Curr Opin Neurobiol 23(6):1065–1072

    CAS  PubMed  Google Scholar 

  19. Peferoen L et al (2014) Oligodendrocyte-microglia cross-talk in the central nervous system. Immunology 141(3):302–313

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Ledeen RW, Golly F, Haley JE (1992) Axon myelin transfer of phospholipids and phospholipid precursors - labeling of myelin phosphoinositides through axonal-transport (molecular neurobiology, vol 6, pg 177, 1992). Mol Neurobiol 6(4):482

    Google Scholar 

  21. Dyer CA (2002) The structure and function of myelin: from inert membrane to perfusion pump. Neurochem Res 27(11):1279–1292

    CAS  PubMed  Google Scholar 

  22. Fields RD et al (2017) Cholinergic signaling in myelination. Glia 65(5):687–698

    PubMed  Google Scholar 

  23. Nagy Z, Westerberg H, Klingberg T (2004) Maturation of white matter is associated with the development of cognitive functions during childhood. J Cogn Neurosci 16(7):1227–1233

    PubMed  Google Scholar 

  24. Deutsch GK et al (2005) Children’s reading performance is correlated with white matter structure measured by diffusion tensor imaging. Cortex 41(3):354–363

    PubMed  Google Scholar 

  25. Liu J et al (2012) Impaired adult myelination in the prefrontal cortex of socially isolated mice. Nat Neurosci 15(12):1621–1623

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Makinodan M et al (2012) A critical period for social experience-dependent oligodendrocyte maturation and myelination. Science 337(6100):1357–1360

    CAS  PubMed  PubMed Central  Google Scholar 

  27. McKenzie IA et al (2014) Motor skill learning requires active central myelination. Science 346(6207):318–322

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Fancy SP et al (2011) Myelin regeneration: a recapitulation of development? Annu Rev Neurosci 34:21–43

    CAS  PubMed  Google Scholar 

  29. Hakak Y et al (2001) Genome-wide expression analysis reveals dysregulation of myelination-related genes in chronic schizophrenia. Proc Natl Acad Sci U S A 98(8):4746–4751

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Najjar S, Pearlman DM (2015) Neuroinflammation and white matter pathology in schizophrenia: systematic review. Schizophr Res 161(1):102–112

    PubMed  Google Scholar 

  31. Tkachev D et al (2003) Oligodendrocyte dysfunction in schizophrenia and bipolar disorder. Lancet 362(9386):798–805

    CAS  PubMed  Google Scholar 

  32. Walterfang M et al (2005) Diseases of white matter and schizophrenia-like psychosis. Aust N Z J Psychiatry 39(9):746–756

    PubMed  Google Scholar 

  33. Yao L et al (2013) White matter deficits in first episode schizophrenia: an activation likelihood estimation meta-analysis. Prog Neuro-Psychopharmacol Biol Psychiatry 45:100–106

    Google Scholar 

  34. Kang SH et al (2013) Degeneration and impaired regeneration of gray matter oligodendrocytes in amyotrophic lateral sclerosis. Nat Neurosci 16(5):571–579

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Philips T, Rothstein JD (2014) Glial cells in amyotrophic lateral sclerosis. Exp Neurol 262:111–120

    CAS  PubMed  Google Scholar 

  36. Aston C, Jiang L, Sokolov BP (2005) Transcriptional profiling reveals evidence for signaling and oligodendroglial abnormalities in the temporal cortex from patients with major depressive disorder. Mol Psychiatry 10(3):309–322

    CAS  PubMed  Google Scholar 

  37. Billiards SS et al (2008) Myelin abnormalities without oligodendrocyte loss in periventricular leukomalacia. Brain Pathol 18(2):153–163

    PubMed  PubMed Central  Google Scholar 

  38. Volpe JJ et al (2011) The developing oligodendrocyte: key cellular target in brain injury in the premature infant. Int J Dev Neurosci 29(4):423–440

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Azzarelli B, Meade P, Muller J (1980) Hypoxic lesions in areas of primary myelination - a distinct pattern in cerebral-palsy. Childs Brain 7(3):132–145

    CAS  PubMed  Google Scholar 

  40. Noseworthy JH et al (2000) Medical progress: multiple sclerosis. N Engl J Med 343(13):938–952

    CAS  PubMed  Google Scholar 

  41. Kuehn BM (2010) Increased risk of ADHD associated with early exposure to pesticides, PCBs. JAMA 304(1):27–28

    CAS  PubMed  Google Scholar 

  42. Sagiv SK et al (2010) Prenatal organochlorine exposure and behaviors associated with attention deficit hyperactivity disorder in school-aged children. Am J Epidemiol 171(5):593–601

    PubMed  PubMed Central  Google Scholar 

  43. Grandjean P, Landrigan PJ (2006) Developmental neurotoxicity of industrial chemicals. Lancet 368(9553):2167–2178

    CAS  PubMed  Google Scholar 

  44. OECD, Test No. 426: Developmental Neurotoxicity Study (2007) OECD Publishing

    Google Scholar 

  45. USEPA, Health effects test guidelines: OPPTS 870.6300, Developmental Neurotoxicity Study [EPA 712–C–98–239]. (1998)

    Google Scholar 

  46. Rice D, Barone S Jr (2000) Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ Health Perspect 108(Suppl 3):511–533

    PubMed  PubMed Central  Google Scholar 

  47. Bal-Price A et al (2018) Strategies to improve the regulatory assessment of developmental neurotoxicity (DNT) using in vitro methods. Toxicol Appl Pharmacol 354:7–18

    CAS  PubMed  PubMed Central  Google Scholar 

  48. EFSA/OECD, Workshop Report on integrated approach for testing and assessment of developmental neurotoxicity (2017) EFSA onlinelibrary

    Google Scholar 

  49. Aschner M et al (2017) Reference compounds for alternative test methods to indicate developmental neurotoxicity (DNT) potential of chemicals: example lists and criteria for their selection and use. Altex 34(1):49–74

    PubMed  Google Scholar 

  50. Fritsche E, Alm H, Baumann J, Geerts L, Håkansson H, Masjosthusmann S, Witters H (2015) Literature review on in vitro and alternative developmental neurotoxicity (DNT) testing methods. EFSA Ext Sci Rep 12(4):778E

    Google Scholar 

  51. Bal-Price A et al (2019) Recommendation on test readiness criteria for new approach methods in toxicology: exemplified for developmental neurotoxicity (vol 35, pg 306, 2018). Altex 36(3):506–506

    PubMed  Google Scholar 

  52. Pamies D et al (2017) A human brain microphysiological system derived from induced pluripotent stem cells to study neurological diseases and toxicity. Altex 34(3):362–376

    PubMed  Google Scholar 

  53. Zhong X, Harris G, Smirnova L, Zufferey V, Baldino Russo F, Baleeiro Beltrao Braga PC, Chesnut M, Zurich MG, Hogberg HT, Hartung T, Pamies D (2020) Antidepressant paroxetine exerts developmental neurotoxicity in an iPSC-derived 3D human brain model. Front Cell Neurosci 14:25

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Hogberg HT et al (2013) Toward a 3D model of human brain development for studying gene/environment interactions. Stem Cell Res Ther 4:1–7

    Google Scholar 

  55. Wen ZX et al (2014) Synaptic dysregulation in a human iPS cell model of mental disorders. Nature 515(7527):414–418

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Skripuletz T et al (2011) De- and remyelination in the CNS white and grey matter induced by cuprizone: the old, the new, and the unexpected. Histol Histopathol 26(12):1585–1597

    CAS  PubMed  Google Scholar 

  57. Matsushima GK, Morell P (2001) The neurotoxicant, cuprizone, as a model to study demyelination and remyelination in the central nervous system. Brain Pathol 11(1):107–116

    CAS  PubMed  Google Scholar 

  58. Torkildsen O et al (2008) The cuprizone model for demyelination. Acta Neurol Scand Suppl 188:72–76

    CAS  PubMed  Google Scholar 

  59. Kerman BE et al (2015) In vitro myelin formation using embryonic stem cells. Development 142(12):2213–2225

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Kuhn S et al (2019) Oligodendrocytes in development, myelin generation and beyond. Cell 8(11):1424

    CAS  Google Scholar 

  61. Zhong X et al (2020) Antidepressant paroxetine exerts developmental neurotoxicity in an iPSC-derived 3D human brain model. Front Cell Neurosci 14:25

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Hartung T, Kavlock R, Sturla SJ (2017) Systems toxicology II: a special issue. Chem Res Toxicol 30(4):869

    CAS  PubMed  Google Scholar 

  63. Hartung T et al (2017) Systems toxicology: real world applications and opportunities. Chem Res Toxicol 30(4):870–882

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Center for Alternatives to Animal Testing (CAAT), Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA

    David Pamies, Megan Chesnut, Thomas Hartung & Helena T. Hogberg

  2. Department of Biomedical Science, University of Lausanne, Lausanne, Switzerland

    David Pamies, Hélène Paschoud & Marie-Gabrielle Zurich

  3. Center for Alternatives to Animal Testing-Europe, University of Konstanz, Konstanz, Germany

    Thomas Hartung

  4. AxoSim, Inc., New Orleans, LA, USA

    Thomas Hartung

  5. Swiss Centre for Applied Human Toxicology (SCAHT), Lausanne, Switzerland

    David Pamies & Marie-Gabrielle Zurich

Authors
  1. David Pamies
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Megan Chesnut
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hélène Paschoud
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marie-Gabrielle Zurich
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Thomas Hartung
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Helena T. Hogberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David Pamies .

Editor information

Editors and Affiliations

  1. Departament de Ciències Fisiològiques, Institut de Neurociènces, Universitat de Barcelona, Hospitalet de Llobregat, Catalunya, Spain

    Jordi Llorens

  2. Departament de Farmacologia, Toxicologia i Química Terapèutica, Universitat de Barcelona, Barcelona, Catalunya, Spain

    Marta Barenys

Ethics declarations

TH, HH, and DP are named inventors on a patent by Johns Hopkins University on the production of mini-brains (also called BrainSpheres), which is licensed to AxoSim, New Orleans, LA, USA. They consult AxoSim and TH is shareholder.

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Pamies, D., Chesnut, M., Paschoud, H., Zurich, MG., Hartung, T., Hogberg, H.T. (2021). Quantification of Oligodendrocytes and Myelin in Human iPSC-Derived 3D Brain Cell Cultures (BrainSpheres). In: Llorens, J., Barenys, M. (eds) Experimental Neurotoxicology Methods. Neuromethods, vol 172. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1637-6_21

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1637-6_21

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1636-9

  • Online ISBN: 978-1-0716-1637-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation